Viral components cross the nuclear membrane occurs at numerous steps in the life cycle of the human immunodeficiency virus (HIV) and other retroviruses. Once in the cytoplasm, HIV RNA is reverse-transcribed into double-stranded DNA which must enter the nucleus. Other retroviral regulatory proteins also enter the nucleus to perform their function, and viral transcripts are exported out to the cytoplasm. The viral proteins, rev and tat, have been identified as a key regulators of the transcription and transport of HIV envelope mRNA out of the nucleus. We have focused on the rev protein of HIV. A chimeric protein has been generated consisting of rev coupled to the green fluorescent protein. An in vitro system using permeabilized cells has been developed to examine the nucleolar targeting of proteins bearing such targeting signals. We have observed that transport to the nucleolus is an active process requiring both GTP and intracellular Ca+2 stores. In addition to its nucleolar transport rev is actively exported from the nucleus. While a specific signal mediating nuclear export of rev has been identified the mechanism of protein export is poorly understood. To examine this nuclear export event we have constructed a chimeric protein consisting of rev coupled to the green fluorescent protein (GFP). The chimera also contains the steroid binding domain of the glucocorticoid receptor thus allowing precise control over nuclear import. Addition of hormone leads to nuclear transport; removal of hormone initiates export but does not allow subsequent import. We have been able to use this nuclear export assay in living cells to determine the requirements for nuclear export. In addition, the assay can be performed in vitro after addition of digitonin to permeabilize the plasma membrane. The assay thus allows identification of soluble factors which are required for nuclear export in vitro.